The EphA4 receptor tyrosine kinase is highly expressed in the developing and adult nervous system, and is upregulated following nervous system injury. A number of important functions have been ascribed to this receptor in neural cells. In the embryonic nervous system, EphA4 interaction with its membrane-associated ligands - the ephrins - are critical for axon guidance as well as for the formation of tissue boundaries. In the adult central nervous system (CNS), EphA4 regulates the structure and plasticity of neuronal connections (synapses) and plays an important role in the communication between glial cells and neurons. For example, we have recently linked EphA4 and one of its ligands, ephrin-A3, to decreased expression of glial glutamate transporters. This suggests that inhibition of EphA4-ephrin-A3 interaction may be beneficial for the treatment of pathologies involving elevated extracellular glutamate levels, such as epilepsy and amyotrophic lateral sclerosis (ALS). In the injured nervous system, EphA4 inhibits nerve regeneration and functional recovery by promoting glial scar formation and inhibiting sprouting of injured axons. Consistent with this, recent studies suggest that inhibiting EphA4-ephrin interaction could be useful for the treatment of spinal cord injuries. Finally, recent work has implicated ephrin-A3 and another EphA4 ligand, ephrin-A2, in maintaining the quiescence of neural precursor cells in the adult brain. This suggests that antagonists of EphA4, which is a major ephrin-A2 and ephrin-A3 receptor in the brain, may be useful for neuronal cell replacement therapies. Thus, EphA4 represents a promising new target for the treatment of CNS disorders. Few molecules that target EphA4 are available. They include small molecule antagonists that target the kinase domain of EphA4 (but also many other tyrosine kinases), and soluble portions of EphA4 and ephrin extracellular domains, which inhibit ephrin binding to EphA4 (but also other Eph receptors). Several peptides and two isomeric small molecules that we identified antagonize EphA4-ephrin binding and are much more selective EphA4 antagonists. Small molecules have more desirable pharmaceutical profiles than peptides as candidates for drug development. However, the two compounds that we have identified and extensively characterized, while highly selective for EphA4 and the related EphA2 receptor, have low potency. In this application, we propose to optimize small molecule antagonists of EphA4-ephrin binding that we identified by high throughput screening. To develop more potent small molecule antagonists, we propose iterations of medicinal chemistry and rapid analoging to generate compounds for testing in biochemical/cell culture assays, and then validate the optimized probe compounds and characterize their activities in neural cells. The optimized EphA4 antagonists will be useful as research tools to obtain valuable insight into the physiological and pathological functions of EphA4, and may also serve as lead compounds for drug development to treat CNS disorders. PUBLIC HEALTH RELEVANCE: EphA4 is a cell surface receptor that plays an important role in the developing and adult nervous system. Evidence suggests that EphA4 inhibits recovery following nervous system injury and may contribute to several nervous system disorders. In this project we propose to improve compounds that we have previously identified and characterized as antagonists of EphA4 function. The optimized antagonists will be useful to further study the physiological and pathological functions of EphA4 and may serve as starting points for drug development to treat disorders of the nervous system.